Prevalence of Coxiella burnetii in cattle at South Korean national ...

RESEARCH ARTICLE

Prevalence of Coxiella burnetii in cattle at South Korean national breeding stock farms Min-Goo Seo1,2, In-Ohk Ouh2, Seung-Hun Lee1,2, Jong Wan Kim2, Man Hee Rhee1, OhDeog Kwon1, Tae-Hwan Kim1, Dongmi Kwak1,3* 1 College of Veterinary Medicine, Kyungpook National University, Daegu, South Korea, 2 Animal and Plant Quarantine Agency, Gimcheon, South Korea, 3 Cardiovascular Research Institute, Kyungpook National University, Daegu, South Korea * [email protected]

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OPEN ACCESS Citation: Seo M-G, Ouh I-O, Lee S-H, Kim JW, Rhee MH, Kwon O-D, et al. (2017) Prevalence of Coxiella burnetii in cattle at South Korean national breeding stock farms. PLoS ONE 12(5): e0177478. https://doi.org/10.1371/journal.pone.0177478 Editor: Kelly A. Brayton, Washington State University, UNITED STATES Received: January 7, 2017 Accepted: April 27, 2017 Published: May 11, 2017 Copyright: © 2017 Seo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information file. Funding: This research was supported by a grant from the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (Grant No. NRF-2013R1A1A2013102). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Abstract This is the first study to evaluate the serologic and molecular prevalence of Coxiella burnetii in cattle at national breeding stock farms in South Korea. These government farms have well-organized biosecurity and management systems to prevent livestock diseases. Of the 736 cattle in this study, 77 tested positive for antibodies against C. burnetii antigens (10.5%, 95% CI: 8.3–12.7) and 11 were positive for a C. burnetti infection on PCR analysis (1.5%, 95% CI: 0.6–2.4). Since the 16S rRNA sequences of C. burnetii from all 11 PCR-positive samples were identical, three representative samples (C-CN-3 from the southern region, C-JJ-9 from Jeju Island, and C-CB-37 from the central region) are described in this paper. These three sequences had 99.3–100% identity to those of C. burnetii deposited in GenBank. These sequences clustered with those from USA, Japan, and Greenland, underscoring the sequence similarity among C. burnetii isolates in these countries. Because C. burnetii was detected in cattle at well-managed national breeding stock farms, cattle at non-government operated farms may be more likely to be exposed to C. burnetii in South Korea. Thus, continuous surveillance and control strategies in animals and humans are required to prevent the transmission of C. burnetii to humans.

Introduction Coxiella burnetii, a zoonotic obligate intracellular bacterium, is the causative agent of Q fever in humans [1]. Q fever has been reported worldwide, except in New Zealand, and differences in host type and host factors affect the prevalence of disease in different regions and countries [1]. Reservoirs include ticks and domestic livestock, which are key sources of C. burnetii transmission [2]. Cattle infected with C. burnetii are usually asymptomatic; however, infection in dairy goats and sheep may result in abortion or stillbirth, often without preceding signs. C. burnetii infection can also cause mastitis, infertility, stillbirth, and reproductive disorders in animals [3], leading to economic losses. The prevalence of C. burnetii infection in ruminants is of concern since Q fever is a zoonotic disease and ruminants are a reservoir for human infection [3]. The bacteria spread

PLOS ONE | https://doi.org/10.1371/journal.pone.0177478 May 11, 2017

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Occurrence and genotypes of Coxiella burnetii in South Korean cattle

Competing interests: The authors have declared that no competing interests exist.

among animals by inhalation of infectious airborne dust or aerosol [4]. Infected animals shed C. burnetii in milk, feces, urine, semen, vaginal mucus, and birth products [3]. Therefore, C. burnetii infection in domestic animals is a public health concern. Dairy farmers, veterinarians, slaughterhouse workers, and anyone regularly interacting with animals or animal products are at risk for exposure to C. burnetii and development of Q fever [5]. Infected aerosols and ingestion of raw milk or dairy products are the primary routes of transmission from animals to humans [6]. National breeding stock farms in South Korea are operated by the government and are geographically isolated from nearby non-government operated farms. There are 17 national breeding stock farms throughout the country. These farms play an important role in improving and enhancing cattle productivity and developing breeding stock. These farms monitor disease and develop control measures to prevent the spread of disease. The national breeding stock farms have a well-organized biosecurity system. Previous studies of the seroprevalence of C. burnetii in cattle at non-government operated farms in Korea [7–9] did not describe the molecular detection of C. burnetii. Therefore, the purpose of this study was to assess the prevalence and genotypes of C. burnetii in cattle at national breeding stock farms in South Korea.

Materials and methods Ethics statement This study, conducted in 2014, did not receive approval from the Institutional Animal Care and Use Committee (IACUC) at Kyungpook National University (KNU), as the IACUC at KNU evaluates laboratory animals maintained in indoor facilities, and not outdoor animals. After receiving consent from the national breeding stock farms, blood samples were collected by practicing veterinarians during treatment or regular medical checkups. This study did not involve endangered or protected species.

Sample size determination and sample collection In 2014, the total number of cattle in South Korea was recorded as 3,189,951 [10]. The sample size was determined using the following formula, with an expected disease prevalence of 50%, an accepted absolute error of 5%, and a confidence level of 99% with a simple random sampling design [11]: n¼

1:962 pexpð1 d2

pexpÞ

where n = required sample size, pexp = expected prevalence, and d = desired absolute precision. The calculated minimum sample size for this study was 663. Samples from 736 cattle were obtained in this study. Cattle were from 17 national breeding stock farms, from nine mainland provinces and Jeju Island, which is located in the southernmost region of South Korea (Fig 1). The farms were well managed in terms of biosecurity, disease, and breeding; however, there were geographic and climatic differences among farms. Blood was obtained from the jugular vein. Whole blood was used for PCR, and serum for serology. Samples were stored at -20˚C until use. Age, sex, breed, and region were recorded for analysis.

Serologic assay Antibodies in serum against C. burnetii were detected using enzyme-linked immunosorbent assay (ELISA) using the ID Screen Q Fever Indirect Multi-species kit (IDvet, Montpellier, France), according to the manufacturer’s instructions. Sensitivity of this kit was 100% (30


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Fig 1. Geographic regions of the national breeding stock farms in South Korea. Blood samples were obtained from cattle at 17 national breeding stock farms from four different regions in Korea. The four regions were the following: Northern [Gyeonggi (GG) and Gangwon (GW)]; Central [Chungbuk (CB), Chungnam (CN), and Gyeongbuk (GB)]; Southern [Jeonbuk (JB), Jeonnam (JN), and Gyeongnam (GN)]; and Jeju Island. The location of the 17 national breeding stock farms are denoted by dots. https://doi.org/10.1371/journal.pone.0177478.g001

bovine serum samples), and specificity was 100% (250 bovine serum and 88 caprine milk samples; IDvet, internal validation report). The ratio of the sample optical density (OD) to the positive control OD (S/P) was calculated for each sample as follows: Value (%) = (OD sample − OD negative control) / (OD positive control − OD negative control) × 100. Samples with S/P greater than 50% were considered positive; between 40% and 50%, doubtful; less than 40%, negative. Samples that were considered doubtful were treated as negative.

Molecular assay Genomic DNA was extracted from the whole blood using a commercial DNeasy Blood and Tissue kit (QIAGEN, Melbourne, Australia), according to the manufacturer’s instructions. The extracted DNA was stored at -20˚C until use. The AccuPower HotStart PCR Premix kit (Bioneer, Daejeon, Korea) was used for PCR amplification. In nested PCR (nPCR), primer sets


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were used to amplify the 16S rRNA of the genus Coxiella, including C. burnetii and Coxiella-like bacteria (CLB) [12, 13]. First-round PCR was performed with the primers Cox16SF1 (5´-CGT AGGAATCTACCTTRTAGWGG-3´) and Cox16SR2 (5´-GCCTACCCGCTTCTGGTACAATT-3´), which produced amplicons of 1321–1429 bp. Then, nPCR was performed using the primers Cox16SF2 (5´-TGAGAACTAGCTGTTGGRRAGT-3´) and Cox16SR2, producing amplicons of 624–627 bp. Samples yielding amplicons of the expected size were bidirectionally sequenced using the primers Cox16SF1 and Cox16SR1 (5´-ACTYYCCAACAGCTAGTTCTCA-3´), which produced amplicons of 719–826 bp. PCR was performed using the Mastercycler Pro (Eppendorf, Hamburg, Germany) with predenaturation at 93˚C for 3 min, followed by 30 cycles of denaturation at 93˚C for 30 s, annealing at 56˚C for 30 s, polymerization at 72˚C for 1 min, and a final post-polymerization at 72˚C for 5 min. After the second amplification, PCR products were separated on 1.5% agarose gels, stained with ethidium bromide, and visualized through UV transillumination.

Sequencing and phylogenetic analysis The purified PCR products were sequenced by Macrogen (Seoul, Korea) using the primers (Cox16SF1 and Cox16SR1) that were used in nPCR. The results were analyzed using the multiple sequence alignment programs, CLUSTAL Omega (ver. 1.2.1), and BioEdit (ver. 7.2.5). Phylogenetic analyses and homology comparisons were performed using MEGA (ver. 6.0) and the maximum-likelihood method. The stability of the phylogenetic tree was estimated using bootstrap analysis with 1,000 replicates.

Statistical analysis The chi-square test was used to analyze differences among the groups. A p-value of < 0.05 was considered statistically significant. The analytical software package GraphPad Prism version 5.04 (GraphPad Software Inc., La Jolla, CA, USA) was used for the statistical analysis. A 95% confidence interval (CI) was calculated for all estimates.

Results Serologic and molecular assays Of the 736 cattle in the study, 77 tested positive for C. burnetii antibodies (10.5%, 95% CI: 8.3– 12.7) and 11 were positive by PCR (1.5%, 95% CI: 0.6–2.4) (Table 1). In addition, nine of 17 farms had C. burnetii antibody positive cattle (52.9%, 95% CI: 29.2–76.7) and five farms had cattle positive by PCR (29.4%, 95% CI: 7.8–51.1). The prevalence of ELISA (p < 0.0001) and PCR (p = 0.0015) positive cattle was significantly associated by herd status (S1 Table). Seroprevalence was significantly higher in cattle on Jeju Island (21.3%, 95% CI: 14.5–28.0) than in any of the other three geographical regions (p < 0.0001). Seroprevalence was significantly higher in black cattle (27.1%, 95% CI: 14.5–39.7) than in any other breed (p < 0.0001). Seropositivity for C. burnetii was significantly higher in female cattle (12.9%, 95% CI: 10.2–15.6) than in male cattle (1.3%, 95% CI: 0–3.1) (p < 0.0001) and significantly increased with age (p < 0.0001). Although the C. burnetii infection rate was low (1.5%; 11/736) when assessed by PCR, the trends with respect to geographical region, breed, sex, and age were similar (Table 1).

DNA sequencing and phylogenetic analysis Results from PCR indicated that 11 animals were positive for C. burnetii infection. Because the sequences of these samples were identical, three samples were used as representative sequences for alignment and phylogenetic analysis. Fig 2 shows a comparative analysis of the nucleotide


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Table 1. Prevalence of Coxiella burnetii in 736 cattle at national breeding stock farms in South Korea during 2014. No. tested

No. of cattle ELISA-positive

95% CIa

p-value

No. of cattle PCR-positive

95% CIa

p-value

Northern

180

8 (4.4)

1.4–7.5

< 0.0001

0

0

0.2041

Central

250

22 (8.8)

5.3–12.3

4 (1.6)

0.1–3.2

Southern

165

17 (10.3)

5.7–14.9

3 (1.8)

0–3.9

Jeju Island

141

30 (21.3)

14.5–28.0

4 (2.8)

0.1–5.6

Brown cattle

523

35 (6.7)

4.6–8.8

6 (1.2)

0.2–2.1

Dairy cattle

155

27 (17.4)

11.5–23.4

3 (1.9)

0–4.1

Black cattle

48

13 (27.1)

14.5–39.7

1 (2.1)

0–6.1

Tiger cattle

10

2 (20.0)

0–44.8

1 (10.0)

0–28.6

Female

581

75 (12.9)

10.2–15.6

Male

155

2 (1.3)

0–3.1